Jessica J. Alm

Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients

We examined the presence of circulating plastic adherent multipotent mesenchymal stem cells (MSCs) in fracture patients. Three patient groups (n = 10–18) were evaluated, including elderly females with a femoral neck fracture treated with cemented hemiarthroplasty, an age‐ and sex‐matched group with hip osteoarthritis (OA) treated with cemented total hip arthroplasty (THA), and younger adults with surgically treated lower extremity fractures. The presence of circulating MSCs pre‐ and postoperatively was compared to bone marrow (BM) MSCs from the same subjects. Criteria for identifying MSCs included cell surface markers (CD105+, CD73+, CD90+, CD45−, CD14−), proliferation through several passages as well as osteogenic, chondrogenic, and adipogenic differentiation. Plastic adherent MSCs were found in peripheral blood (PB) from 22% of hip fracture patients, 46% of younger fracture patients, and in none of 63 pre‐ and postmenopausal women with hip OA. When detectable, circulating MSCs appeared between 39 and 101 h after fracture. PB derived MSCs did not differ from BM derived MSCs, except for a small population (<15%) of CD34+ cells among PB derived MSCs. This initial study indicates mobilization of MSCs into the circulation in response to fracture, even in very old patients, while circulating MSCs were not detectable before or after elective THA.

MicroRNAs miR‐96, miR‐124, and miR‐199a regulate gene expression in human bone marrow‐derived mesenchymal stem cells

MicroRNAs are small non‐coding RNAs that control gene expression at the post‐transcriptional level by binding to 3′‐untranslated regions (3′‐UTR) of their target mRNAs. They present a promising tool to delineate the molecular mechanisms regulating differentiation of human mesenchymal stromal cells (hMSCs) and to improve the controlled differentiation of hMSCs in therapeutic applications. Here we show that three microRNAs, miR‐96, miR‐124, and miR‐199a, were differentially expressed during osteogenic, adipogenic, and chondrogenic induction of human bone marrow‐derived MSCs. miR‐96 expression was increased during osteogenesis and adipogenesis, but not during chondrogenesis. miR‐124 was exclusively expressed in adipocytes, whereas miR‐199a was upregulated in osteoblasts and chondrocytes. Furthermore, functional studies with synthetic miRNA precursors and inhibitors demonstrated that miR‐96, miR‐124, and miR‐199a regulated the expression of genes important for hMSC differentiation, such as aggrecan, transcription factor SOX9, and fatty acid binding protein 4 (FABP4). Modulation of miR‐96, miR‐124, and miR‐199a expression may thus be useful in specific targeting of hMSC differentiation, for e.g., MSC‐based therapies. J. Cell. Biochem. 113: 2687–2695, 2012.

Low BMD affects initial stability and delays stem osseointegration in cementless total hip arthroplasty in women: a 2-year RSA study of 39 patients.

Background and purpose Immediate implant stability is a key factor for success in cementless total hip arthroplasty (THA). Low bone mineral density (BMD) and age-related geometric changes of the proximal femur may jeopardize initial stability and osseointegration. We compared migration of hydroxyapatite-coated femoral stems in women with or without low systemic BMD. Patients and methods 61 female patients with hip osteoarthritis were treated with cementless THA with anatomically designed hydroxyapatite-coated femoral stems and ceramic-ceramic bearing surfaces (ABG-II). Of the 39 eligible patients between the ages of 41 and 78 years, 12 had normal systemic BMD and 27 had osteopenia or osteoporosis. According to the Dorr classification, 21 had type A bone and 18 had type B. Translational and rotational migration of the stems was evaluated with radiostereometric analysis (RSA) up to 2 years after surgery. Results Patients with low systemic BMD showed higher subsidence of the femoral stem during the first 3 months after surgery than did those with normal BMD (difference = 0.6, 95% CI: 0.1–1.1; p = 0.03). Low systemic BMD (odds ratio (OR) = 0.1, CI: 0.006–1.0; p = 0.02), low local hip BMD (OR = 0.3, CI: 0.1–0.7; p = 0.005) and ageing (OR = 1.1, CI: 1.0–1.2; p = 0.02) were risk factors for delayed translational stability. Ageing and low canal flare index were risk factors for delayed rotational stabilization (OR = 3, CI: 1.1–9; p = 0.04 and OR = 1.1, CI: 1.0–1.2; p = 0.02, respectively). Harris hip score and WOMAC score were similar in patients with normal systemic BMD and low systemic BMD. Interpretation Low BMD, changes in intraosseous dimensions of the proximal femur, and ageing adversely affected initial stability and delayed osseointegration of cementless stems in women.

Female patients with low systemic BMD are prone to bone loss in Gruen zone 7 after cementless total hip arthroplasty

Background and purpose Factors that lead to periprosthetic bone loss following total hip arthroplasty (THA) may not only depend on biomechanical implant-related factors, but also on various patient-related factors. We investigated the association between early changes in periprosthetic bone mineral density (BMD) and patient-related factors. Patients and methods 39 female patients underwent cementless THA (ABG II) with ceramic-ceramic bearing surfaces. Periprosthetic BMD in the proximal femur was determined with DXA after surgery and at 3, 6, 12, and 24 months. 27 patient-related factors were analyzed for their value in prediction of periprosthetic bone loss. Results Total periprosthetic BMD was temporarily reduced by 3.7% at 3 months (p < 0.001), by 3.8% at 6 months (p < 0.01), and by 2.6% at 12 months (p < 0.01), but recovered thereafter up to 24 months. Preoperative systemic osteopenia and osteoporosis, but not the local BMD of the operated hip, was predictive of bone loss in Gruen zone 7 (p = 0.04), which was the only region with a statistically significant decrease in BMD (23%, p < 0.001) at 24 months. Preoperative serum markers of bone turnover predicted the early temporary changes of periprosthetic BMD. The other patient-related factors failed to show any association with the periprosthetic BMD changes. Interpretation Female patients with low systemic BMD show greater bone loss in Gruen zone 7 after cementless THA than patients with normal BMD. Systemic DXA screening for osteoporosis in postmenopausal patients before THA could be used to identify patients in need of prophylactic anti-resorptive therapy.

Quality of intertrochanteric cancellous bone as predictor of femoral stem RSA migration in cementless total hip arthroplasty

In cementless total hip arthroplasty, osteoporosis may jeopardize the achievement of immediate stability and lead to migration of anatomically shaped femoral stems. Poor quality of proximal cancellous bone per se may also affect the rate of osseointegration. In a selected group of female total hip arthroplasty patients (mean age 64 years) with unremarkable medical history, intertrochanteric cancellous bone biopsy was taken from the site of stem implantation. Local bone quality, determined by structural μCT imaging and destructive compression testing of the biopsy tissue, was used as the predictor of three-dimensional stem migration determined by radiostereometric analysis (RSA) up to 24 months. The patients exhibited major differences in mechanical properties of the intertrochanteric cancellous bone, which were closely related to the structural parameters calculated from μCT data. Unexpectedly, the major differences observed in the quality of trochanteric cancellous bone had only minor reflections in the RSA migration of the femoral stems. In statistical analysis, the μCT-based bone mineral density quartile (low, middle, high) was the only significant predictor for stem translation at 24 months (p=0.022) but only a small portion (R(2)=0.16) of the difference in translation could be explained by changes in bone mineral density quartile. None of the other parameters investigated predicted stem migration in translation or rotation. In conclusion, poor quality of intertrochanteric cancellous bone seems to contribute to the risk of implant migration less than expected. Probably also the importance of surgical preservation of intertrochanteric cancellous bone has been over-emphasized for osseointegration of cementless stem.

Comparison of the osteogenic capacity of minipig and human bone marrow-derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made.

In vivo testing of a biodegradable woven fabric made of bioactive glass fibers and PLGA80--a pilot study in the rabbit.

The purpose of this study was to perform an intra-animal comparison of biodegradable woven fabrics made of bioactive glass (BG) fibers and poly(L-lactide-co-glycolide) 80/20 copolymer (PLGA(80)) fibers or PLGA(80) fibers alone, in surgical stabilization of bone graft. The BG fibers (BG 1-98) were aimed to enhance bone growth at site of bone grafting, whereas the PLGA component was intended to provide structural strength and flexibility to the fabric. Bone formation was analyzed qualitatively by histology and quantitatively by peripheral quantitative computed tomography (pQCT) at 12 weeks. The surgical handling properties of the control PLGA(80) fabric were more favorable. Both fabrics were integrated with the cortical bone surfaces, but BG fibers showed almost complete resorption. There were no signs of adverse local tissue reactions. As a proof of material integration and induced new bone formation, there was a significant increase in bone volume of the operated femurs compared with the contralateral intact bone (25% with BG/PLGA(80) fabric, p < 0.001 and 28% with the control PLGA(80) fabric, p = 0.006). This study failed to demonstrate the previously seen positive effect of BG 1-98 on osteogenesis, probably due to the changed resorption properties of BG in the form of fibers. Therefore, the feasibility and safety of BG as fibers needs to be reevaluated before use in clinical applications.

Increased migration of uncemented acetabular cups in female total hip arthroplasty patients with low systemic bone mineral density - A 2-year RSA and 8-year radiographic follow-up study of 34 patients

Background and purpose — Low bone mineral density (BMD) may jeopardize the initial component stability and delay osseointegration of uncemented acetabular cups in total hip arthroplasty (THA). We measured the migration of uncemented cups in women with low or normal BMD. Patients and methods — We used radiostereometric analysis (RSA) to measure the migration of hydroxyapatite-coated titanium alloy cups with alumina-on-alumina bearings in THA of 34 female patients with a median age of 64 (41–78) years. 10 patients had normal BMD and 24 patients had low systemic BMD (T-score ≤ −1) based on dual-energy X-ray absorptiometry (DXA). Cup migration was followed with RSA for 2 years. Radiographic follow-up was done at a median of 8 (2–10) years. Results — Patients with normal BMD did not show a statistically significant cup migration after the settling period of 3 months, while patients with low BMD had a continuous proximal migration between 3 and 12 months (p = 0.03). These differences in cup migration persisted at 24 months. Based on the perceived risk of cup revision, 14 of the 24 cases were “at risk” (proximal translation of 0.2 to 1.0 mm) in the low-BMD group and 2 of the10 cases were “at risk” in the normal-BMD group (odds ratio (OR) = 8.0, 95% CI: 1.3–48). The radiographic follow-up showed no radiolucent lines or osteolysis. 2 cups have been revised for fractures of the ceramic bearings, but none for loosening. Interpretation — Low BMD contributed to cup migration beyond the settling period of 3 months, but the migrating cups appeared to osseointegrate eventually.

Enhanced osteoblastic differentiation and bone formation in co-culture of human bone marrow mesenchymal stromal cells and peripheral blood mononuclear cells with exogenous VEGF

BACKGROUND Despite recent advances in bone tissue engineering, efficient bone formation and vascularization remains a challenge for clinical applications. HYPOTHESIS The aim of this study was to investigate if the osteoblastic differentiation of human mesenchymal stromal cells (MSCs) can be enhanced by co-culturing them with peripheral blood (PB) mononuclear cells (MNCs), with and without vascular endothelial growth factor (VEGF), a coupling factor of bone formation and angiogenesis. MATERIALS AND METHODS Human bone marrow (BM) derived MSCs were co-cultured with PB-MNCs in osteogenic medium with or without VEGF. Osteoblastic differentiation and mineral deposition were studied by staining for alkaline phosphatase (ALP), and von Kossa, respectively, and measurements for ALP activity and calcium concentration (Ca). Cell proliferation was assayed with Alamar blue. The mechanism(s) were further studied by Transwell(®) cell culture experiments. RESULTS Both ALP and mineralization (von Kossa and Ca) were significantly higher in the MSC-MNC co-cultures compared to plain MSC cultures. VEGF alone had no effect on osteoblastic differentiation of MSCs, but further enhanced differentiation in co-culture settings. The mechanism was shown to require cell-cell contact between MSCs and MNCs and the factors contributing to further differentiation appear to be soluble. No differences were observed in cell proliferation. CONCLUSION Our study demonstrates that the in vitro ALP activity and mineralization of human BM-MSCs is more efficient in the presence of PB-MNCs, and exogenously added VEGF further enhances the stimulatory effect. This indicates that PB-MNCs could be a potential cell source in development of co-culture systems for novel tissue engineering applications for enhanced bone healing.

Interaction between Marrow-Derived Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells in Endothelial Cell Differentiation

Background and Aims: In adult connective tissues, mesenchymal stem cells (MSCs) play a key role in normal tissue turnover and repair. MSCs can participate in these processes not only through proliferation and differentiation but also through paracrine/autocrine functions. These characteristics make MSCs the optimal target in the development of cell-based therapies. This study describes a novel interaction between human MSC and blood mononuclear cells (MNCs), resulting in formation of blood vessel-like structures. Materials and Methods: Human marrow-derived MSCs and peripheral blood MNCs were co-cultured in monolayer cultures as well as in bovine collagen sponge up to 20 days. No exogenously supplied growth factors were applied. Morphological changes and formations of three dimensional structures were detected by light microscopy. The process was further studied for the expression of different endothelial cell markers. The expression of PECAM-1 and endoglin was studied by immunohistochemistry and the expression of vascular endothelial growth factor receptors 1 and 2 using quantitative real time PCR. Results: In co-cultures of human MSCs and MNCs, the previously nonadherent cells attached and started to elongate and formed tube-like structures within one week. At day 10, elongated PECAM-1 and endoglin expressing cells were detected in co-cultures. At day 20, PECAM-1 and endoglin-positive vessel-like structures were observed. VEGFR1 was up-regulated in co-cultures after 10 days, and expression levels increased with time. No PECAM-1, endoglin or VEGFR1 expressing cells were discovered in MSC-cultures without MNCs at any time point. Conclusions: This study demonstrates induction of endothelial differentiation in co-cultures of human MSCs and MNCs, indicating a mechanism by which local application of MSCs could induce angiogenesis in vivo.